Cardiomyocyte phenotype and mechanotransduction in Filamin C gene variants causing arrhythmogenic cardiomyopathy Project Summary For over two decades, our laboratories have investigated the genetic basis of cardiomyopathies, heart muscle diseases that are a major cause of morbidity and mortality in the world. Recently, we discovered a novel cardiomyopathy disease gene, filamin C (FLNC), and noted that truncating loss-of-function variants (FLNCtv) in FLNC lead to arrhythmogenic cardiomyopathy (ACM), characterized by a high risk of life-threatening ventricular arrhythmias and progression to heart failure. However, FLNC function is still poorly understood and significant knowledge gaps preclude therapeutic development. Notably, (i) the cellular localization and interactions of FLNC, in particular at the cell-cell junction, are incompletely resolved, (ii) the spectrum of molecular networks involved in filaminopathies is largely unknown, (iii) the biomechanical properties of cardiomyocytes with mutant FLNC are also unknown, (iv) the role of FLNC in sarcomere function is not completely elucidated, and (v) finally, the mechanism by which FLNC variants cause different clinical phenotypes is unknown. This proposal aims to determine mechanisms of myocardial failure and cardiac arrhythmia in FLNCtv. Our overarching hypothesis is that FLNCtv perturb mechanotransduction machinery due to disruption of the sarcomeric cytoskeleton, resulting in stress signaling pathway activation (integrins/hippo pathway) which in turn triggers fibrogenesis and adipogenesis, ultimately providing the substrate for arrhythmia. To address these gaps, we have generated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from FLNCtv patients and from CRISPR/Cas9-edited lines, collected frozen explanted hearts from FLNCtv patients, and gathered a multidisciplinary research team experienced in experimental modeling of cardiomyopathies. Based on a series of proof-of-concept experiments and preliminary data, we propose three Specific Aims: Aim 1. Determine the phenotype and mechanisms of functional impairment and electrical dysfunction in FLNCtv. We will determine the mechanisms of structural and functional alterations, changes in electrophysiological function, and dysregulation of the interactome at the sarcomere-cytoskeletal-desmosomal interface in hiPSC-CMs. Aim 2. Identify the mechanisms of altered biomechanics in FLNCtv human hearts and hiPSC-CMs. We will determine the mechanisms of altered biomechanics by single cell spectroscopy and myofibrillar mechanics of mutant FLNC hiPSC-CMs and explanted hearts of FLNCtv patients. Aim 3. Define the mechanism of gene expression dysregulation in FLNCtv cardiomyopathy. Using cardiac tissue from FLNCtv patients and FLNC hiPSC-CM models, we will assess role of altered mechanosignaling (Hippo/YAP, TGF?, Wnt), discover novel transcriptional changes in FLNCtv heart tissue and hiPSC-CMs models, and provide the mechanistic link with structural, contractile and electrophysiological alterations. The elucidation of molecular networks activated in FLNCtv will provide the mechanistic link with the structural, contractile and electrophysiological alterations, and lay the foundation for targeted rescue experiments.